Engineering Salmonella typhimurium to be a targeted anti-cancer therapeutic Bacteria engineered to specifically target therapeutically resistant regions of tumors and controllably kill cancer cells will be able to overcome therapeutic resistance and increase the efficacy of cancer treatment. Therapeutic resistance is caused by limited drug penetration and poor cell susceptibility. Only motile bacteria, which can penetrate into tumor tissue and overcome diffusion limitations, are able to effectively kill therapeutically resistant cells distant from tumor vasculature. Controlling bacterial motility is therefore the key to developing effective therapies able to overcome therapeutic resistance. To date, the mechanisms of bacterial motility in tumors are poorly understood. Our approach will elucidate these mechanisms and will manipulate them to target tumor quiescence. The project is unique because it introduces the concept of intratumoral therapeutic delivery and the cylindroid tumor model, which was specifically designed to quantify bacterial chemotaxis in tumors. The proposed research program has three Specific Aims that combine expertise in tumor biology, molecular biology, and mathematical modeling. They are interrelated and will be performed concurrently: Aim 1 is to determine the mechanisms that control S. typhimurium accumulation in tumors;Aim 2 is to design S. typhimurium mutants with enhanced targeting;and Aim 3 is to create S. typhimurium mutants with increased tumor cytotoxicity. The three hypotheses of the research plan are: 1) S. typhimurium are attracted to and induce cellular apoptosis in tumors, 2) ribose-receptor-knockout S. typhimurium preferentially accumulate in quiescent regions of tumors, and 3) S. typhimurium expressing a radiation-inducible, cytotoxic polypeptide payload will more effectively reduce tumor mass. These hypotheses will be tested by measuring the localization of S. typhimurium in mouse tumors, deleting the ribose receptor from the S. typhimurium genome, and transforming S. typhimurium to express the mTRAIL polypeptide under control of the RecA promoter. The efficiency of the transformed bacteria to reduce tumor mass will be measured in culture and in mice. The experimental plan is part of a research program to develop a therapeutic strategy to overcome therapeutic resistance. Combined administration of tumor-quiescence-targeted S. typhimurium and adjuvant chemotherapy will increase therapeutic efficiency by more effectively killing cancer cells distant from tumor vasculature. Future human trials will investigate the ability of combined administration of bacteria and chemotherapy to reduce local recurrence and metastatic disease in stage-four breast cancer patients. Using bacteria to overcome therapeutic resistance and increase treatment efficiency will significantly reduce systemic toxicity, limit the deleterious effects of metastatic disease, and increase life expectancy. The experimental plan descries a therapeutic strategy to overcome therapeutic resistance using quiescence- targeted, controllably cytotoxic S. typhimurium. Combined administration of engineered bacteria and adjuvant chemotherapy will increase therapeutic efficiency by more effectively killing all cancer cells in tumors. This increased efficacy will reduce systemic toxicity, prevent local recurrence, limit the deleterious effects of metastatic disease, and increase life expectancy.